Zhonghua Minguo wei sheng wu ji mian yi xue za zhi = Chinese journal of microbiology and immunology最新文献

Mutants of Bacillus thuringiensis subsp. kurstaki NTU 9 and Bt 158, which were isolated previously for using the diamondback moth as a target insect in Taiwan, were screening by either protein electrophoresis of intracellular proteins or enzyme-linked immunosorbent assay (ELISA). The optimal conditions of effective protein electrophoresis were (1) 24-hour cells harvested from nutrient broth were crashed by petite glass beads followed by centrifugation. And (2) the supernatant pretreated by heating at 60 degrees C for 2 minutes was electrophoresed with 7.5% native PAGE at 110 voltages. On ELISA, the antiserum used was obtained from rabbits immunized with Bt 158 crystal protein. Optimal antigen coating concentration of ELISA, attained by chequer-board titration method, was 10 micrograms/ml. Antigens (crystal protein) in samples were detected by competitive inhibition method with antiserum diluted to 10(4) fold. By using protein electrophoresis and ELISA methods, two isolates A 71 and BN 11, were denoted respectively as qualitative and quantitative mutants of Bacillus thuringiensis.

Immobilized cell technology has been playing a vital role in the development of fermentation processes. For the past several years, I have been working on immobilized cell systems with an aim of developing novel immobilized biosystems where physical, chemical as well as biological functions are incorporated into the immobilization carrier. By efficiently integrating these new functions with the innate abilities of immobilized cells, the area where immobilized cell systems can be utilized will expand, and the process efficiency will be greatly improved.

A total of 273 serum specimens from different areas and sources were tested against Borrelia burgdorferi antigens by enzyme-linked immunosorbent assay method. Positive rates of serological reactions were 3% and 58% for healthy persons and syphilis patients, respectively. Obviously, there was a lot of cross-reaction in the venereal disease group. Meanwhile, positive rates were 3% and 13% in the sera collected from Taiwan and Orchid Island, respectively. This difference may reflect a less developed environment in the latter. Since reported cases of Lyme disease in Taiwan are rare, serologic tests are usually adapted for rapid diagnosis in common laboratories. As for disease confirmation, clinical observations, epidemiological data and exposure in an endemic area must also be considered.

The transposon derivative has been placed on a transposition suicide vector to yield pDB30 in Escherichia coli WA803. A simple method, using a Tn5 derivative Tn5-Lux, has been successfully devised for the introduction and stable expression of the bioluminescence property in Pseudomonas sp., Agrobacterium sp., and Rhizobium sp. In this study, there was also successful mating between Escherichia coli WA803(pDB30) and strains of Acromonas hydrophila and Plesiomonas shigelloides. These bacteria emitted bioluminescence after they gained pDB30 by transconjugation.

Two proteolytic proteins (about 43 and 90 kDa) were produced by clinical strains of Vibrio parahaemolyticus cultured in iron-limited medium. The 43 kDa-protease was partially purified by ammonium sulfate precipitation, ultrafiltration fractionation and DEAE-Sephacel chromatography. This protease had an optimum pH range of 7 to 8, and an optimum reaction temperature of about 40 degrees C. It was heat-labile, being partially inactivated by heat-treatment at 60 or 90 degrees C for 10 min. The protease hydrolyzed casein, gelatin, elastin, collagen and hemoglobin. As a chymotrypsin-like protease, it was inhibited only by the chymostatin among seven protease inhibitors tested. Activity of this protease was partially inhibited by 1 mM of Co2+, Cu2+, Zn2+ and Hg2+ and slightly enhanced by Ca2+ and Ba2+. It was completely inactivated by orthophenanthroline (OPA), and the OPA-inactivated sample was partially reactivated by Ca2+ and Fe2+. In conclusion, this 43-kDa protease of V. parahaemolyticus was an unstable neutral chymotrypsin-like metalloprotease; Ca2+ and/or Fe2+ was essential for its activity or stability.

Clindamycin is one of the antimicrobial agents most commonly used against anaerobes. Resistance to clindamycin in Bacteroides fragilis has been increasing recently. Thirty strains of clindamycin-resistant (including multi-resistant) B. fragilis were collected for study of cross-resistance to beta-lactam agents and beta-lactam--beta-lactamase inhibitor and resistance transferability. Imipenem was the most active drug against these 30 isolates. Resistance to clindamycin was transferred to a recipient in 12 out of 30 donor strains by using filter-mating. Of 12 transconjugants, only three had detectable plasmids by alkaline lysis method and the remaining nine strains lacked plasmids. The transfer frequencies ranged from 10(-4) to 10(-6). The role of plasmid in the resistance transfer was not certain. However, the results suggest that non-plasmid-mediated transfer accounted for the majority of the transfers of clindamycin-resistance of B. fragilis in this study. Tetracycline resistance was co-transferred from six donors. There was no evidence of co-transference of beta-lactam resistance under the selection marker of clindamycin, beta-lactam, or both. Therefore, non-plasmid-mediated transfer may play an important role in dissemination of resistance transfer in B. fragilis in Taiwan.

The bactericidal/permeability-increasing protein (BPI) of polymorphonuclear leukocytes is a potent antibacterial agent specific for gram-negative bacteria. BPI can bind to lipopolysaccharide (LPS) and neutralize its toxicity. However, little is known about the specific site and mechanisms of the BPI involved in this LPS binding and antibacterial activities. This study compared the amino acid sequences among BPI, cecropin A, magainin 2, and polymyxin B, and identified a common structure among these four bactericidal agents. They share a basic amphipathic alpha helix motif (Baah). A short peptide that represents amino acids 90-101 of BPI was then synthesized to test if it possessed any LPS binding and antibacterial activities. Results from in vitro lymphocyte culture indicated this peptide was able to inhibit LPS-induced lymphocyte proliferation, suggesting that it may interact with LPS. This LPS binding ability of BPI peptide 90-101 was further supported by the results from HPLC assays which showed the mobility of the peptide shifted in the presence of LPS. Furthermore, the antibacterial spectra of this peptide and cecropin peptide 1-11 were very similar to that of polymyxin B, even though the antibacterial activities of these two peptides were less potent than that of polymyxin B. In addition, the antibacterial activities of these two peptides and polymyxin B were inhibited by free LPS or a high concentration of MgCl2. These results thus suggest that a common structure (Baah) and antibacterial mechanism may be involved in these antibacterial agents.

The methylviologen-dependent hydrogenase of Anabaena sp. CH3 is unstable at 4 degrees C and in air. All the purifying procedures were carried out at 25 degrees C and under argon atmosphere. The enzyme was partially purified by the following steps: heat treatment, DEAE-cellulose ion-exchange chromatography and gel filtration on TSK-Fractogel. Experimental results indicated that the heat-treatment procedure was beneficial for the separation of the enzyme from phycobiliproteins.

A rapid isoniazid (INH) susceptibility test was developed for Mycobacterium tuberculosis by using acridinium-ester (AE)-labeled DNA probe. The method was applied to two reference strains and 20 clinical isolates, then compared with the standard proportion method. By comparing the difference of log relative light units (RLUs) M. tuberculosis cultures incubating in the presence and absence of INH, INH susceptibility could be determined with the AE-DNA probe after three to five days of incubation. The difference of log RLUs of susceptible strains was statistically significantly lower than that for resistant strains after incubation for three to five days. The cutoff value was defined as "mean + one standard deviation" of the difference between log RLU(INH) and log RLU(no INH) on Day 5. By this criterion, agreement between the AE-DNA probe and the proportion concentration method was found in 19 of 20 susceptibility tests (95%). The AE-DNA probe test is rapid and non-isotopic, and may provide a useful alternative method for INH susceptibility testing for clinical isolates of M. tuberculosis.

Allopurinol hypersensitivity syndrome (AHS) is an infrequent but life-threatening adverse reaction of allopurinol therapy. The records of 38 patients with the allopurinol hypersensitivity syndrome evaluated at the Veterans General Hospital-Taipei were reviewed. The clinical pictures included fever, rash, leukocytosis, eosinophilia, impaired renal function and hepatocellular injury. Nine patients died (24%) and the major cause of death was infection. The use of corticosteroids increased neither survival nor mortality rate. Twenty-six percent of patients were treated with allopurinol for asymptomatic hyperuricemia, which was not an established indication of the drug, should be avoided. The most important factor of mortality was toxic epidermal necrolysis (TEN) (p < 0.001 compared with other skin lesions). As there is no way to identify the risk group of patients or to make effective treatment for AHS, the only means of minimizing the incidence of AHS is to limit the allopurinol therapy to accepted indications and to adjust the dosage for the patient's renal function.